Adjuvant nanoemulsions with crystallisation inhibitors

ABSTRACT

High loading of lipophilic pharmacological agents (in particular, lipophilic immunopotentiators) in oil-in-water emulsions can result in crystallisation of the lipophilic agent. To overcome this problem the invention uses an oil-in-water emulsion in combination with a crystallisation inhibitor, and this combination provides emulsions which can be loaded with high levels of lipophilic pharmacological agents.

This application is the U.S. National Phase of International ApplicationNo. PCT/US2012/022871, filed Jan. 27, 2012 and published in English,which claims the benefit of U.S. Provisional Application No. 61/436,946,filed on Jan. 27, 2011. The entire contents of the foregoingapplications are incorporated herein by reference.

TECHNICAL FIELD

The invention is in the field of emulsions which are useful fordelivering lipophilic pharmacological agents, such asimmunopotentiators.

BACKGROUND ART

Pharmacological agents can require formulation in order to optimisetheir in vivo effects. For instance, they might be encapsulated oradsorbed. Appropriate formulation can provide, for instance, homogeneousdosing, improved efficacy, better pharmacokinetics, or simplermanufacturing. For example, reference 1 encapsulated indomethacin inpolymeric nanoparticles, and reference 2 formulated a lipophilic muramyldipeptide immunopotentiator with polylactide microspheres.

It is an object of the invention to provide further and improved ways offormulating lipophilic pharmacological agents for in vivo use, includingmethods which offer improved processing during manufacture when comparedto simple monodispersions of the lipophilic agent.

DISCLOSURE OF THE INVENTION

The inventors attempted to formulate lipophilic pharmacological agents(in particular, lipophilic immunopotentiators) in oil-in-wateremulsions, but the emulsions could accommodate only low concentrationsof agent. Higher loading led to crystallisation of the lipophilic agent,which gives an undesirably unstable emulsion. To overcome this observedcrystallisation the invention uses an oil-in-water emulsion incombination with a crystallisation inhibitor, and this combinationprovides emulsions which can be loaded with high levels of lipophilicpharmacological agents.

Thus the invention provides an oil-in-water emulsion comprising anaqueous phase, an oil phase, a surfactant, and a crystallisationinhibitor. These emulsions are useful for formulating lipophilicpharmacological agents for in vivo use, and so the invention alsoprovides an oil-in-water emulsion comprising an aqueous phase, an oilphase, a surfactant, a crystallisation inhibitor, and a lipophilicpharmacological agent.

The invention also provides an immunogenic composition comprising (a) anoil-in-water emulsion of the invention and (b) an immunogen.

The invention also provides a process for preparing an oil-in-wateremulsion, comprising a step of homogenising a mixture comprising anaqueous component, an oil component, and a surfactant component, whereina crystallisation inhibitor is added to the mixture before, during orafter homogenisation (preferably before). A lipophilic pharmacologicalagent may be added (i) to the mixture before during or afterhomogenisation, or (ii) to the emulsion before or after addition of thecrystallisation inhibitor; it is preferably added to the mixture beforehomogenisation and before addition of the crystallisation inhibitor. Thehomogenisation may comprise microfluidisation.

The invention also provides a process for preparing an immunogeniccomposition, comprising a step of mixing an immunogen with an emulsionof the invention.

Lipophilic Pharmacological Agents

Emulsions of the invention are useful for formulating lipophilicpharmacological agents (LPAs) for in vivo use. Such LPAs include, butare not limited to, vitamins (e.g. vitamins A, D, E and K and theirsalts/esters, such as α-tocopherol succinate), carotenoids (e.g.β-carotene), fatty acids (e.g. arachidonic acid, ecosapentaenoic aciddocosahexaenoic acid), pyrimidines (e.g.5-hydroxy-4,6-dimethyl-2-(6-phenylhexyl)aminopyrimidine), ansamitocins,angiotensin II receptor antagonists (e.g. candesartan cilexetil), andimmunopotentiators (e.g. muramyl dipeptides). Typical LPAs have apositive log P value (partition coefficient measured in 1-octanol andwater) at pH 7.4 and 37° C. e.g. they may have a log P value ≥1, ≥2, ≥3,≥4, ≥5, ≥6, ≥7, etc.

The invention is particularly useful for formulating LPAs which cancrystallise. Thus it is useful for LPAs that crystallise when present inan oil-in-water emulsion which is identical to an emulsion of theinvention except for absence of the crystallisation inhibitor (i.e. thesame aqueous phase, oil phase, surfactant and LPA as an emulsion of theinvention, in the same amounts, but lacking the crystallisationinhibitor).

Preferred LPAs for use with the invention are small molecule immunepotentiators (SMIPs). These SMIPs have a molecular weight of less than5000 Da (e.g. <4000 Da, <3000 Da, <2000 Da, or <1000 Da). They mayfunction as agonists of one or more of human toll-like receptors TLR1,TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and/or TLR11, or mayagonise other receptors such as CD1d. Lipophilic agonists of TLR1include lipopeptides. Lipophilic agonists of TLR2 include glycolipidsand lipoteichoic acid.

Lipophilic agonists of TLR7 include the naphthyridines disclosed inreference 3. Specific lipophilic SMIPs for use with the inventioninclude, but are not limited to, the compounds disclosed in the examplesof reference 3 e.g. example 161 (compound ‘A’ herein). Compound ‘A’ arevery lipophilic and highly crystalline.

Emulsions of the invention may include only one LPA or may includemultiple LPAs.

In preferred emulsions, most or all (e.g. ≥80%, ≥85%, ≥90%, ≥95%) of thetotal LPA is present (dissolved) in the emulsion's oil droplets.

The concentration of LPA in an emulsion of the invention can vary over awide range e.g. between 10 μg/ml to 50 mg/ml, between 0.1 mg/ml to 5mg/ml, or between 0.5 mg/ml to 2 mg/ml.

Crystallisation Inhibitors

Emulsions of the invention include a crystallisation inhibitor. Varioussuch inhibitors are known in the art. For instance, an emulsion of theinvention may include a polyvinylpyrrolidone (PVP), ahydroxypropylmethylcellulose (HPMC), a poloxamer, a dextrin derivative,a polyethylene glycol (PEG), a polypropylene glycol (PPG), a polyvinylacetate (PVA), a copolymer of vinylpyrrolidone and vinyl acetate, orglycerin [4-6]. Preferred crystallisation inhibitors are polymers.

PVP is a water-soluble polymer formed from N-vinylpyrrolidone monomers.Useful PVPs can have a K value (Fikentscher's viscosity coefficient)between 10-150 e.g. between 12-100, between 12-20, or between 15-30. Oneuseful PVP has a K value of 15.

Useful HPMCs include salts or esters such as hydroxypropylmethylcellulose acetate succinate.

Poloxamers are non-ionic copolymers with a central hydrophobic chain ofpolyoxypropylene flanked by two hydrophilic chains of polyoxyethylene.Useful poloxamers for crystallisation inhibition include poloxamer “407”(Lutrol 127) [6].

Useful crystallisation inhibitors are typically amorphous e.g. PVP.

The amount of a crystallisation inhibitor in an emulsion of theinvention can vary widely but will depend on the LPA in question and itsdesired dose. The amount of crystallisation inhibitor in the emulsionwill be enough to ensure that substantially all of the LPA remainsamorphous for the emulsion's desired shelf life with an adequate LPAloading.

Emulsions of the invention may include only one crystallisationinhibitor or may include multiple crystallisation inhibitors.

The crystallisation inhibitor will usually be located in the emulsion'soil phase, although it may also be present in the aqueous phase.

Oil-in-water Emulsions

Emulsions of the invention comprise oil droplets in an aqueous bulkphase. The emulsions include a surfactant and this can facilitateformation and stabilisation of the emulsion.

The emulsion may comprise one or more oils and/or fatty acids. Suitableoil(s) include those from, for example, an animal (such as fish) or avegetable source. The oil is ideally biodegradable (metabolisable) andbiocompatible. Sources for vegetable oils include nuts, seeds andgrains. Peanut oil, soybean oil, coconut oil, and olive oil, the mostcommonly available, exemplify the nut oils. Jojoba oil can be used e.g.obtained from the jojoba bean. Seed oils include safflower oil,cottonseed oil, sunflower seed oil, sesame seed oil and the like. In thegrain group, corn oil is the most readily available, but the oil ofother cereal grains such as wheat, oats, rye, rice, teff, triticale andthe like may also be used. 6-10 carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and so may be used. The procedures forseparation, purification, saponification and other means necessary forobtaining pure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalane, the saturated analog to squalene, can also be used. Fish oils,including squalene and squalane, are readily available from commercialsources or may be obtained by methods known in the art.

Other useful oils are the tocopherols, particularly in combination withsqualene. Where the oil phase of an emulsion includes a tocopherol, anyof the α,β, γ, δ, ε or ξ tocopherols can be used, but α-tocopherols arepreferred. D-α-tocopherol and DL-α-tocopherol can both be used. Apreferred α-tocopherol is DL-α-tocopherol. An oil combination comprisingsqualene and a tocopherol (e.g. DL-α-tocopherol) can be used.

Preferred emulsions comprise squalene, a shark liver oil which is abranched, unsaturated terpenoid (C₃₀H₅₀; [(CH₃)₂C[═CHCH₂CH₂C(CH₃)]₂═CHCH₂—]₂;2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN7683-64-9).

The oil in the emulsion may comprise a combination of oils e.g. squaleneand at least one further oil.

The aqueous component of the emulsion can be plain water (e.g. w.f.i.)or can include further components e.g. solutes. For instance, it mayinclude salts to form a buffer e.g. citrate or phosphate salts, such assodium salts. Typical buffers include: a phosphate buffer; a Trisbuffer; a borate buffer; a succinate buffer; a histidine buffer; or acitrate buffer. A buffered aqueous phase is preferred, and buffers willtypically be included in the 5-20 mM range.

The surfactant in the emulsion is preferably biodegradable(metabolisable) and biocompatible. Surfactants can be classified bytheir ‘HLB’ (hydrophile/lipophile balance), where a HLB in the range1-10 generally means that the surfactant is more soluble in oil than inwater, and a HLB in the range 10-20 are more soluble in water than inoil. Emulsions preferably comprise at least one surfactant that has aHLB of at least 10 e.g. at least 15, or preferably at least 16.

The invention can be used with surfactants including, but not limitedto: the polyoxyethylene sorbitan esters surfactants (commonly referredto as the Tweens), especially polysorbate 20 and polysorbate 80;copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butyleneoxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO blockcopolymers; octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); polyoxyethylene fatty ethersderived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brijsurfactants), such as triethyleneglycol monolauryl ether (Brij 30);polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known asthe Spans), such as sorbitan trioleate (Span 85) and sorbitanmonolaurate. Preferred surfactants for including in the emulsion arepolysorbate 80 (Tween 80; polyoxyethylene sorbitan monooleate), Span 85(sorbitan trioleate), lecithin and Triton X-100.

Mixtures of surfactants can be included in the emulsion e.g. Tween80/Span 85 mixtures, or Tween 80/Triton-X100 mixtures. A combination ofa polyoxyethylene sorbitan ester such as polyoxyethylene sorbitanmonooleate (Tween 80) and an octoxynol such ast-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable.Another useful combination comprises laureth-9 plus a polyoxyethylenesorbitan ester and/or an octoxynol. Useful mixtures can comprise asurfactant with a HLB value in the range of 10-20 (e.g. polysorbate 80,with a HLB of 15.0) and a surfactant with a HLB value in the range of1-10 (e.g. sorbitan trioleate, with a HLB of 1.8).

In addition to the oil, aqueous and surfactant components, an emulsioncan include further components. For instance, an emulsion can includecholesterol and/or a phospholipid. Inclusion of a phospholipid canincrease the loading capacity of an emulsion. Suitable classes ofphospholipid include, but are not limited to, phosphatidylethanolamines,phosphatidylcholines, phosphatidylserines, phosphatidylglycerols, etc.Useful phospholipids are listed in Table 1. These phospholipidcomponents will typically be present in an emulsion's oil phase.

Preferred amounts of oil (% by volume) in the final emulsion are between2-20% e.g. 5-15%, 6-14%, 7-13%, 8-12%. A squalene content of about 4-6%or about 9-11% is particularly useful.

Preferred amounts of surfactants (% by weight) in the final emulsion arebetween 0.001% and 8%. For example: polyoxyethylene sorbitan esters(such as polysorbate 80) 0.2 to 4%, in particular between 0.4-0.6%,between 0.45-0.55%, about 0.5% or between 1.5-2%, between 1.8-2.2%,between 1.9-2.1%, about 2%, or 0.85-0.95%, or about 1%; sorbitan esters(such as sorbitan trioleate) 0.02 to 2%, in particular about 0.5% orabout 1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100)0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers(such as laureth 9) 0.1 to 8%, preferably 0.1 to 10% and in particular0.1 to 1% or about 0.5%.

The absolute amounts of oil and surfactant, and their ratio, can bevaried within wide limits while still forming an emulsion. A skilledperson can easily vary the relative proportions of the components toobtain a desired emulsion, but a weight ratio of between 4:1 and 5:1 foroil and surfactant is typical (excess oil). The amounts of cholesteroland/or phospholipid can similarly be varied widely while obtaining adesired emulsion e.g. between 0.5 mg/ml and 20 mg/ml e.g. between 2-10mg/ml, or between 3-7 mg/ml, or about 5 mg/ml. The amounts of thesevarious components may be selected to provide a useful formulation ofthe relevant LPA e.g. to ensure that there is sufficient oil to dissolvethe desired dose of LPA, etc., while giving a stable emulsion with highLPA loading.

An important parameter for ensuring immunostimulatory activity of anemulsion, particularly in large animals, is the oil droplet size(diameter). The most effective emulsions have a droplet size in thesubmicron range. Suitably the droplet sizes will be in the range 50-750nm. Most usefully the average droplet size is less than 250 nm e.g. lessthan 200 nm, less than 150 nm. The average droplet size is usefully inthe range of 80-180 nm. Ideally, at least 80% (by number) of theemulsion's oil droplets are less than 250 nm in diameter, and preferablyat least 90%. Apparatuses for determining the average droplet size in anemulsion, and the size distribution, are commercially available. Thesethese typically use the techniques of dynamic light scattering and/orsingle-particle optical sensing e.g. the Accusizer™ and Nicomp™ seriesof instruments available from Particle Sizing Systems (Santa Barbara,USA), or the Zetasizer™ instruments from Malvern Instruments (UK), orthe Particle Size Distribution Analyzer instruments from Horiba (Kyoto,Japan).

Ideally, the distribution of droplet sizes (by number) has only onemaximum i.e. there is a single population of droplets distributed aroundan average (mode), rather than having two maxima. Preferred emulsionshave a polydispersity of <0.4 e.g. 0.3 or less.

Suitable emulsions with submicron droplets and a narrow sizedistribution can be obtained by the use of microfluidisation. Thistechnique reduces average oil droplet size by propelling streams ofinput components through geometrically fixed channels at high pressureand high velocity. These streams contact channel walls, chamber wallsand each other. The results shear, impact and cavitation forces cause areduction in droplet size. Repeated steps of microfluidisation can beperformed until an emulsion with a desired droplet size average anddistribution are achieved.

As an alternative to microfluidisation, thermal methods can be used tocause phase inversion, as disclosed in reference 7. These methods canalso provide a submicron emulsion with a tight particle sizedistribution.

Preferred emulsions can be filter sterilised i.e. their droplets canpass through a 220 nm filter. As well as providing a sterilisation, thisprocedure also removes any large droplets in the emulsion.

Preferred emulsions are adjuvant emulsions i.e. they can provide an invivo immunostimulatory effect in a mammal even if administered withoutthe lipophilic agent and without the crystallisation inhibitor. Knownadjuvant emulsions, in which a lipophilic agent and a crystallisationinhibitor can be incorporated, include:

-   -   An emulsion comprising squalene, polysorbate 80 (Tween 80), and        sorbitan trioleate (Span 85). The composition of the emulsion by        volume can be about 5% squalene, about 0.5% polysorbate 80 and        about 0.5% sorbitan trioleate. In weight terms, these amounts        become 4.3% squalene, 0.5% polysorbate 80 and 0.48% sorbitan        trioleate. This adjuvant is known as ‘MF59’. The MF59 emulsion        advantageously includes citrate ions e.g. 10 mM sodium citrate        buffer.    -   Emulsions comprising squalene, an α-tocopherol (ideally        DL-α-tocopherol), and polysorbate 80. These emulsions may have        (by weight) from 2 to 10% squalene, from 2 to 10% α-tocopherol        and from 0.3 to 3% polysorbate 80 e.g. 4.3% squalene, 4.7%        α-tocopherol, 1.9% polysorbate 80. The weight ratio of        squalene:tocopherol is preferably ≤1 (e.g. 0.90) as this        provides a more stable emulsion. Squalene and polysorbate 80 may        be present volume ratio of about 5:2, or at a weight ratio of        about 11:5. One such emulsion can be made by dissolving        polysorbate 80 in PBS to give a 2% solution, then mixing 90 ml        of this solution with a mixture of (5 g of DL-α-tocopherol and 5        ml squalene), then microfluidizing the mixture. The resulting        emulsion may have submicron oil droplets e.g. with an average        diameter of between 100 and 250 nm, preferably about 180 nm.    -   An emulsion comprising squalene, a polysorbate (e.g. polysorbate        80), a Triton detergent (e.g. Triton X-100) and a tocopherol        (e.g. an α-tocopherol succinate). The emulsion may include these        three components at a mass ratio of about 75:11:10 (e.g. 750        μg/ml polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include a 3d-MPL. The emulsion may also include a        saponin, such as QS21. The aqueous phase may contain a phosphate        buffer.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [7]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. It may also        include a TLR4 agonist, such as one whose chemical structure        does not include a sugar ring [8]. Such emulsions may be        lyophilized.        Immunogens

Where the LPA is an immune potentiator, emulsions of the invention areuseful for co-delivery with immunogens, thereby providing enhancedimmunogenicity. Thus an immunogenic composition of the invention cancomprise a SMIP-containing emulsion of the invention and an immunogen.

The immunogen may elicit an immune response against a bacterium, avirus, a fungus or a parasite. As an alternative to eliciting an immuneresponse against a pathogen, the immunogen may be a self antigen forimmunotherapy e.g. a cancer antigen.

Bacterial immunogens can comprise proteins, saccharides, and/orlipopolysaccharides. They may be live bacteria, inactivated bacteria, orbacterial subunits. Examples of useful immunogens elicit an immuneresponse against:

-   -   Neisseria meningitidis: useful immunogens include, but are not        limited to, membrane proteins and/or capsular saccharides.        Capsular saccharides from serogroups A, C, W135, and/or Y are        useful. Adhesins, autotransporters, toxins, iron acquisition        proteins, and factor H binding proteins are useful membrane        protein immunogens. A preferred vaccine includes the protein        antigens disclosed in reference 9.    -   Streptococcus pneumoniae: useful immunogens include, but are not        limited to, proteins and/or capsular saccharides. For example,        capsular saccharides from any of pneumococcal serotypes 1, 2, 3,        4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,        19A, 19F, 20, 22F, 23F, and/or 33F can be used.    -   Streptococcus pyogenes: useful immunogens include, but are not        limited to, proteins and/or capsular saccharides. Useful        proteins are disclosed in references 10 and 11.    -   Moraxella catarrhalis.    -   Bordetella pertussis: Useful pertussis immunogens include, but        are not limited to, pertussis toxin or toxoid (PT), filamentous        haemagglutinin (FHA), pertactin, and agglutinogens 2 and 3.    -   Staphylococcus aureus: Useful immunogens include, but are not        limited to, proteins and/or capsular saccharides. For example,        type 5 and/or type 8 capsular saccharides can be used.    -   Clostridium tetani: The typical immunogen is tetanus toxoid.    -   Cornynebacterium diphtheriae: the typical immunogen is        diphtheria toxoid.    -   Haemophilus influenzae type B: the typical Hib immunogen is its        capsular saccharide, PRP.    -   Pseudomonas aeruginosa    -   Streptococcus agalactiae: useful immunogens include, but are not        limited to, proteins and/or capsular saccharides. Useful        proteins are disclosed in reference 12. Capsular saccharides        from one or more of serotypes Ia, Ib, Ia/c, II, III, IV, V, VI,        VII and VIII can be used.    -   Chlamydia trachomatis: Useful immunogens include, but are not        limited to, PepA, LcrE, ArtJ, DnaK, CT398, OmpH-like, L7/L12,        OmcA, AtoS, CT547, Eno, HtrA and MurG (e.g. as disclosed in        reference 13. LcrE [14] and HtrA [15] are two preferred        immunogens.    -   Chlamydia pneumoniae: Useful immunogens include, but are not        limited to, the proteins disclosed in reference 16.    -   Helicobacter pylori: Useful immunogens include, but are not        limited to, CagA, VacA, NAP, and/or urease [17].    -   Escherichia coli: Useful immunogens include, but are not limited        to, immunogens derived from enterotoxigenic E. coli (ETEC),        enteroaggregative E. coli (EAggEC), diffusely adhering E. coli        (DAEC), enteropathogenic E. coli (EPEC), extraintestinal        pathogenic E. coli (ExPEC) and/or enterohemorrhagic E. coli        (EHEC). ExPEC strains include uropathogenic E. coli (UPEC) and        meningitis/sepsis-associated E. coli (MNEC). Useful UPEC        immunogens are disclosed in references 18 and 19. Useful MNEC        immunogens are disclosed in reference 20. A useful immunogen for        several E. coli types is AcfD [21].    -   Bacillus anthracia    -   Yersinia pestis: Useful immunogens include, but are not limited        to, those disclosed in references 22 and 23.    -   Salmonella typhi: the typical S. typhi immunogen is its capsular        saccharide, Vi.

Where the immunogen is a saccharide, it will usually be conjugated to acarrier protein. For example, pneumococcal, Hib, S. aureus, S. typhi andmeningococcal saccharide conjugate vaccines are known in the art.Carrier proteins are typically a bacterial toxin or toxoid (e.g. adiphtheria or tetanus toxoid, or a non-toxic mutant form thereof e.g.CRM197 [24]), but other carriers can be used. For example, suitablecarrier proteins include but are not limited to: N. meningitidis outermembrane protein complex [25], synthetic peptides [26,27], heat shockproteins [28,29], pertussis proteins [30,31], cytokines [32],lymphokines [32], hormones [32], growth factors [32], artificialproteins comprising multiple human CD4⁺ T cell epitopes from variouspathogen-derived antigens [33] such as N19 [34], protein D from H.influenzae [35-37], iron-uptake proteins [38], toxin A or B from C.difficile [39], recombinant P. aeruginosa exoprotein A (rEPA) [40],pneumolysin [41] or its non-toxic derivatives [42], pneumococcal surfaceprotein PspA [43], etc.

Viral immunogens can comprise proteins. They may be live viruses,inactivated viruses, or viral subunits. Examples of useful immunogenselicit an immune response against:

-   -   Orthomyxovirus: Useful immunogens can be from an influenza A, B        or C virus. A live attenuated virus or an inactivated virus can        be used, including a whole inactivated virus, a split virus, or        viral surface glycoproteins (including hemagglutinin). The        vaccine may be monovalent, 2-valent, 3-valent, 4-valent or more.    -   Paramyxoviridae viruses: Viral immunogens include, but are not        limited to, those derived from Pneumoviruses (e.g. respiratory        syncytial virus), Paramyxoviruses (e.g. parainfluenza virus),        Metapneumoviruses and Morbilliviruses (e.g. measles).    -   Poxyiridae: Viral immunogens include, but are not limited to,        those derived from Orthopoxvirus such as Variola vera, including        but not limited to, Variola major and Variola minor.    -   Picornavirus: Viral immunogens include, but are not limited to,        those derived from Picornaviruses, such as Enteroviruses,        Rhinoviruses, Heparnavirus, Cardioviruses and Aphthoviruses. In        one embodiment, the enterovirus is a poliovirus e.g. a type 1,        type 2 and/or type 3 poliovirus.    -   Bunyavirus: Viral immunogens include, but are not limited to,        those derived from an Orthobunyavirus, such as California        encephalitis virus, a Phlebovirus, such as Rift Valley Fever        virus, or a Nairovirus, such as Crimean-Congo hemorrhagic fever        virus.    -   Heparnavirus: Viral immunogens include, but are not limited to,        those derived from a Heparnavirus, such as hepatitis A virus        (HAV).    -   Togavirus: Viral immunogens include, but are not limited to,        those derived from a Togavirus, such as a Rubivirus, an        Alphavirus, or an Arterivirus. This includes rubella virus.    -   Flavivirus: Viral immunogens include, but are not limited to,        those derived from a Flavivirus, such as Tick-borne encephalitis        (TBE) virus, Dengue (types 1, 2, 3 or 4) virus, Yellow Fever        virus, Japanese encephalitis virus, Kyasanur Forest Virus, West        Nile encephalitis virus, St. Louis encephalitis virus, Russian        spring-summer encephalitis virus, Powassan encephalitis virus.    -   Pestivirus: Viral immunogens include, but are not limited to,        those derived from a Pestivirus, such as Bovine viral diarrhea        (BVDV), Classical swine fever (CSFV) or Border disease (BDV).    -   Hepadnavirus: Viral immunogens include, but are not limited to,        those derived from a Hepadnavirus, such as Hepatitis B virus. A        composition can include hepatitis B virus surface antigen        (HBsAg).    -   Other hepatitis viruses: A composition can include an immunogen        from a hepatitis C virus, delta hepatitis virus, hepatitis E        virus, or hepatitis G virus.    -   Rhabdovirus: Viral immunogens include, but are not limited to,        those derived from a Rhabdovirus, such as a Lyssavirus (Rabies        virus) and Vesiculovirus (VSV).    -   Caliciviridae: Viral immunogens include, but are not limited to,        those derived from Calciviridae, such as Norwalk virus, and        Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain        Virus.    -   Coronavirus: Viral immunogens include, but are not limited to,        those derived from a SARS coronavirus, avian infectious        bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine        transmissible gastroenteritis virus (TGEV). The coronavirus        antigens may comprise spike protein.    -   Retrovirus: Viral immunogens include, but are not limited to,        those derived from an Oncovirus, a Lentivirus or a Spumavirus.    -   Reovirus: Viral immunogens include, but are not limited to,        those derived from an Orthoreovirus, a Rotavirus, an Orbivirus,        or a Coltivirus.    -   Parvovirus: Viral immunogens include, but are not limited to,        those derived from Parvovirus B19.    -   Herpesvirus: Viral immunogens include, but are not limited to,        those derived from a human herpesvirus, such as, by way of        example only, Herpes Simplex Viruses (HSV), Varicella-zoster        virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV),        Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and        Human Herpesvirus 8 (HHV8).    -   Papovaviruses: Viral immunogens include, but are not limited to,        those derived from Papillomaviruses and Polyomaviruses. The        Papillomavirus may be of serotype 1, 2, 4, 5, 6, 8, 11, 13, 16,        18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g. from        one or more of serotypes 6, 11, 16 and/or 18.    -   Adenovirus: Viral immunogens include those derived from        adenovirus serotype 36 (Ad-36).

Fungal immunogens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme; or fromAspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi,Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus,Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata,Candida krusei, Candida parapsilosis, Candida stellatoidea, Candidakusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis,Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis,Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum,Histoplasma capsulatum, Klebsiella pneumoniae, Microsporidia,Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi;the less common are Brachiola spp, Microsporidium spp., Nosema spp.,Pleistophora spp., Trachipleistophora spp., Vittaforma sppParacoccidioides brasiliensis, Pneumocystis carinii, Pythiumninsidiosum, Pityrosporum ovale, Sacharomyces cerevisae, Saccharomycesboulardii, Saccharomyces pombe, Scedosporium apiosperum, Sporothrixschenckii, Trichosporon beigelii, Toxoplasma gondii, Penicilliummarneffei, Malassezia spp., Fonsecaea spp., Wangiella spp., Sporothrixspp., Basidiobolus spp., Conidiobolus spp., Rhizopus spp, Mucor spp,Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp.,Alternaria spp, Curvularia spp, Helminthosporium spp, Fusarium spp,Aspergillus spp, Penicillium spp, Monolinia spp, Rhizoctonia spp,Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

Emulsion Formation

Emulsions of the invention can be made by various methods. As mentionedabove, microfluidisation or phase inversion can be used to provideemulsions with small oil droplets. Ideally the emulsion's components arecombined before these techniques are used e.g. such that all ofcomponents are microfluidised together. In other embodiments, though, anemulsion may be formed from the aqueous, surfactant and oil components,and then the LPA and crystallisation inhibitor can be added. Usually theLPA and crystallisation inhibitor are added to the oil, and this oilymixture is combined with the aqueous component prior tomicrofluidisation, with surfactant being added either as a thirdcomponent or as part of the oily or aqueous component. Other orders ofmixing can also be used. In general, an immunogen will be added after anemulsion is formed e.g. after microfluidisation.

One method for forming the emulsion comprises combining aqueous, oil andsurfactant components with an organic solution of the LPA e.g. in avolatile organic solvent such as dichloromethane or methylene chloride.The volatile solvent can then be evaporated e.g. after homogenisation ofthe mixture. After evaporation the homogenised mixture can bemicrofluidised.

Pharmaceutical Compositions and Products

Emulsions and immunogenic compositions of the invention are for in vivouse (in humans or animals) and so should include only pharmaceuticallyacceptable components. Useful pharmaceutical components, such ascarrier(s) and/or excipient(s), are discussed in reference 44.

Pharmaceutical compositions may include one or more preservatives, suchas thiomersal or 2-phenoxyethanol. Mercury-free compositions arepreferred, and preservative-free vaccines can be prepared.

Pharmaceutical compositions can have an osmolality of between 200mOsm/kg and 400 mOsm/kg, e.g. between 240-360 mOsm/kg, or between290-310 mOsm/kg.

Pharmaceutical compositions typically have a pH between 5.0 and 9.5 e.g.between 6.0 and 8.0.

Pharmaceutical compositions are preferably sterile.

Pharmaceutical compositions preferably non-pyrogenic e.g. containing <1EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EUper dose.

Pharmaceutical compositions are preferably gluten free.

Pharmaceutical compositions of the invention may be prepared in unitdose form. In some embodiments a unit dose may have a volume of between0.1-1.0 ml e.g. about 0.5 ml.

The compositions may be prepared as injectables e.g. for intramuscularinjection.

Methods of Treatment and Medical Uses

The invention provides a method for raising an immune response in amammal comprising the step of administering an effective amount of animmunogenic composition of the invention. The immune response ispreferably protective and preferably involves antibodies and/orcell-mediated immunity. The method may raise a booster response.

The invention also provides an emulsion or immunogenic composition ofthe invention for use in raising an immune response in a mammal.

The invention also provides the use of an emulsion or immunogeniccomposition of the invention in the manufacture of a medicament forraising an immune response in a mammal.

By raising an immune response in the mammal by these uses and methods,the mammal can be protected against various diseases and/or infectionse.g. against bacterial and/or viral diseases as discussed above.

The invention also provides a delivery device containing an immunogeniccomposition of the invention. This device can be used to administer thecomposition to a mammalian subject.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably a teenager or an adult. A vaccine intended for children mayalso be administered to adults e.g. to assess safety, dosage,immunogenicity, etc.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or mucosally, such as byrectal, oral (e.g. tablet, spray), vaginal, topical, transdermal ortranscutaneous, intranasal, ocular, aural, pulmonary or other mucosaladministration.

The invention may be used to elicit systemic and/or mucosal immunity,preferably to elicit an enhanced systemic and/or mucosal immunity.

Preferably the enhanced systemic and/or mucosal immunity is reflected inan enhanced TH1 and/or TH2 immune response. Preferably, the enhancedimmune response includes an increase in the production of IgG1 and/orIgG2a and/or IgA.

Dosage can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Multiple doses will typically be administered at least 1 week apart(e.g. about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.). In oneembodiment, multiple doses may be administered approximately 6 weeks, 10weeks and 14 weeks after birth, e.g. at an age of 6 weeks, 10 weeks and14 weeks, as often used in the World Health Organisation's ExpandedProgram on Immunisation (“EPI”). In an alternative embodiment, twoprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the second primary dose, e.g. about 6, 8, 10 or 12months after the second primary dose. In a further embodiment, threeprimary doses are administered about two months apart, e.g. about 7, 8or 9 weeks apart, followed by one or more booster doses about 6 monthsto 1 year after the third primary dose, e.g. about 6, 8, 10, or 12months after the third primary dose.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Thus a human patient may be less than 1 year old,less than 5 years old, 1-5 years old, 5-15 years old, 15-55 years old,or at least 55 years old. Preferred patients for receiving the vaccinesare the elderly (e.g. ≥50 years old, ≥60 years old, and preferably ≥65years), the young (e.g. ≤5 years old), hospitalised patients, healthcareworkers, armed service and military personnel, pregnant women, thechronically ill, or immunodeficient patients. The vaccines are notsuitable solely for these groups, however, and may be used moregenerally in a population.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows % recovery of compound ‘A’ agonist in emulsions ANE12 toANE18, both before (hatched) and after (white) centrifugation.

FIG. 2 shows microscope images of emulsions (A) ANE05 (B) ANE31 (C)ANE32 and (D) MF59.

FIG. 3 shows levels of compound ‘A’ in (A) liver, (B) muscle and (C)serum. Values are in μM.

FIG. 4 shows cytokine levels over 24 hours for animals receivingdifferent formulations of compound ‘A’. The cytokines are (A) IFNγ (B)IL10 and (C) TNF.

FIG. 5 shows HPLC recovery of compound ‘A’ from (A) ANE03, (B) ANE31 and(C) ANE32. For each pair of bars the left is recovery at time zero andthe right after 7 days at room temperature.

MODES FOR CARRYING OUT THE INVENTION

Reference 3 discloses a series of TLR7 agonist naphthyridines, includingcompound ‘A’ (example 161 on page 269):

Although these compounds are highly lipophilic (e.g. log P of compound‘A’ is 7.5) they are not soluble in oil at concentrations which would bedesirable for in vivo use. For example, to deliver a 25 μg dose in atypical oil-in-water emulsion would require the agonist to have asqualene solubility of 10 mg/ml, which is about 2.5× higher than can beachieved.

Lipids were added to the MF59 emulsion in an attempt to increase itscapacity for solubilising the agonists. A variety of differentphospholipid chain lengths (POPC, DLPC, DMPC, DSPC; emulsions “ANE06” to“ANE10”) were tested at a 25 μg dose of agonist, and also cholesterol.These emulsions were visually stable, and provided high loading(essentially 100%), but after storage at room temperature the particlesize was observed to grow. Further investigation revealed that theagonist was crystallising within the emulsion. Emulsions ANE12 to ANE18were tested by HPLC for agonist content before and after centrifugationto separate small and large droplets. A lower value after centrifugationindicates that the agonist was dissolved in larger unstable droplets. Asshown in FIG. 1, although the addition of lipids increased loadingcompared to ANE12, recovery dropped for all of the emulsions, indicatingthat the extra agonist was crystallising and causing emulsioninstability.

As the emulsion was unstable, various attempts were made to preventcrystallization of the agonist within the oil phase. PVP was added as acrystallisation inhibitor, at various amounts from 2.5 mg/ml up to 10mg/ml (ANE19 to ANE25). Loading was high (≥80%) and agonist content wassubstantially the same before and after centrifugation i.e. the PVP waspreventing crystallisation of the agonist.

Emulsions ANE05 and ANE19 to ANE24 were stored at room temperature for aweek to accelerate any crystallisation which might occur. The content ofcompound ‘A’ dropped by about 15% in the control emulsion ANE05, but wasstable in the other emulsions, showing that PVP prevents crystallisationfor at least a week.

Emulsions ANE31 to ANE38 were used to study the effect of addingphospholipid (DSPC) or cholesterol together with PVP. ANE32 (DSPC andPVP) had the highest loading before and after 1 week at roomtemperature. PVP alone increased stability but did not increase loading(ANE33).

FIG. 2 shows emulsions ANE05, ANE31 and ANE32 after 1 week at roomtemperature, compared to MF59 (2D). ANE31 is more disperse than ANE05,but aggregates are still present. In contrast, ANE32 with DSPC and PVPis visibly more stable.

FIG. 5 shows HPLC recovery of compound ‘A’ from ANE05, ANE31 and ANE32.Recovery is both higher and more stable in ANE32.

The following table summarises the effect of the various componentsadded to the starting emulsion:

+lipid “Blank” +lipid +cholesterol +cholesterol Emulsion +lipid+cholesterol +PVP +PVP +PVP +PVP Loading ~70% ~100% ~100% ~80% ~95% ~85%~85% Stable No No No Yes Yes Yes Yes

Thus addition of phospholipids or cholesterol (5 mg/ml) increasedloading of the emulsions but did not improve stability (assessed bydroplet size and by microscopy). Addition of PVP improved stability andgave a small increase in loading. Addition of cholesterol and PVPfurther increased loading, and very high loading was seen with acombination of phospholipid and PVP.

Pharmacokinetics

Levels of compound ‘A’ were assessed in liver and muscle for five groupsof mice: (a) simple monodispersion of compound ‘A’; (b) monodispersionand separately administered emulsion; (c) ANE05; (d) ANE31; and (e)ANE32. As shown in FIG. 3A, liver levels are the highest in group (a),and then fall through (b) to (e), but are very low in all groups. Musclelevels (FIG. 3B) are higher in groups (a) and (b) than in groups (c) to(e). Incorporation of compound ‘A’ in the emulsion's oil phase thusseems to clear the drug from the muscle.

Serum levels were also monitored (FIG. 3C). After 24 hours levels forgroups (a) and (b) are higher than for (c) to (e) but are again verylow.

Cytokine profiles were also studied. Animals received 25 μg compound ‘A’as (a) a monodispersion (c) as a monodispersion with MF59 (c) as ANE05or (d) as ANE32. The profile for ANE32 was markedly different for IFN-γ,IL10, IL12p40, IL1b, IL5, IL6, MCP and TNF, although the levels for allgroups were low and so comparisons are difficult. FIG. 4 shows threeexample profiles.

Immunogenicity Studies

The serogroup B meningococcus vaccine of reference 9 was tested withemulsion ANE32 as adjuvant in CD1 mice. For comparison, the sameantigens were also tested with MF59, with a monodispersion of compound‘A’ combined with MF59 or ANE38. ANE32 gave the highest antibody titerat day 28 (2432), but it was not significantly higher than the ‘A’+MF59group (2284). Both were higher than for the monodispersion with ANE38(1509) or for MF59 alone (873). Although the ANE32 titer was not betterthan the ‘A’+MF59 group, the emulsion was more stable for an extendedperiod and so is advantageous e.g. it can be filter-sterilised, thussimplifying manufacture.

Emulsion Details and Characterisation

Specific emulsions which were tested are as follows, with a 20 mlvolume:

ANE05 4.3% sq, 0.5% S85, 0.5% T80, 10 mg ‘A’ ANE06 4.3% sq, 0.5% S85,0.5% T80, 10 mg ‘A’, 100 mg chol ANE07 4.3% sq, 0.5% S85, 0.5% T80, 10mg ‘A’, 100 mg chol, 100 mg POPC ANE08 4.3% sq, 0.5% S85, 0.5% T80, 10mg ‘A’, 100 mg chol, 100 mg DLPC ANE09 4.3% sq, 0.5% S85, 0.5% T80, 10mg ‘A’, 100 mg chol, 100 mg DMPC ANE10 4.3% sq, 0.5% S85, 0.5% T80, 10mg ‘A’, 100 mg chol, 100 mg DSPC ANE11 4.3% sq, 0.5% S85, 0.5% T80, 4 mg‘A’ ANE12 2.15% sq, 0.25% S85, 0.25% T80, 10 mg ‘A’ ANE13 2.15% sq,0.25% S85, 0.25% T80, 10 mg ‘A’, 100 mg DLPC ANE14 2.15% sq, 0.25% S85,0.25% T80, 10 mg ‘A’, 100 mg chol, 100 mg DLPC ANE15 2.15% sq, 0.25%S85, 0.25% T80, 10 mg ‘A’, 100 mg DMPC ANE16 2.15% sq, 0.25% S85, 0.25%T80, 10 mg ‘A’, 100 mg chol, 100 mg DMPC ANE17 2.15% sq, 0.25% S85,0.25% T80, 10 mg ‘A’, 100 mg DSPC ANE18 2.15% sq, 0.25% S85, 0.25% T80,10 mg ‘A’, 100 mg chol, 100 mg DSPC ANE19 4.3% sq, 0.5% S85, 0.5% T80,10 mg ‘A’, 100 mg DLPC, 50 mg PVP ANE20 4.3% sq, 0.5% S85, 0.5% T80, 10mg ‘A’, 100 mg chol, 100 mg DLPC, 50 mg PVP ANE21 4.3% sq, 0.5% S85,0.5% T80, 10 mg ‘A’, 100 mg DLPC, 100 mg PVP ANE22 4.3% sq, 0.5% S85,0.5% T80, 10 mg ‘A’, 100 mg chol, 100 mg DLPC, 100 mg PVP ANE23 4.3% sq,0.5% S85, 0.5% T80, 10 mg ‘A’, 100 mg DLPC, 200 mg PVP ANE24 4.3% sq,0.5% S85, 0.5% T80, 10 mg ‘A’, 100 mg chol, 100 mg DLPC, 200 mg PVPANE31 4.3% sq, 0.5% S85, 0.5% T80, 10 mg ‘A’, 100 mg DSPC ANE32 4.3% sq,0.5% S85, 0.5% T80, 10 mg ‘A’, 100 mg DSPC, 50 mg PVP ANE33 4.3% sq,0.5% S85, 0.5% T80, 10 mg ‘A’, 50 mg PVP ANE34 4.3% sq, 0.5% S85, 0.5%T80, 10 mg ‘A’, 100 mg DSPC, 50 mg PVP, 100 mg chol ANE35 4.3% sq, 0.5%S85, 0.5% T80, 10 mg ‘A’, 50 mg PVP, 100 mg chol T80 = Tween 80; S85 =Span 85; chol = cholesterol; sq = squalene

These emulsions were generally prepared as follows. Lipophiliccomponents (squalene, span 85, phospholipids, compound ‘A’) werecombined in a beaker. Lipid components were dissolved in chloroform,tetrahydrofuran or dichloromethane. The oil phase was combined with theaqueous phase and immediately homogenized for 2 minute using an IKA T25homogenizer at 24K RPM in order to provide a homogeneous feedstock.Emulsions were immediately emulsified and then allowed to sit at roomtemperature on a stirplate for 2-3 hours after primary homogenization ina fume hood. The primary emulsions were passed three to five timesthrough a Microfluidizer M110S homogenizer with an ice bath cooling coilat a homogenization pressure of approximately 15 k-20 k PSI(Microfluidics, Newton, Mass.). The 20 ml batch samples were removedfrom the unit and stored at 4° C., and 5 ml aliquots were stored at roomtemperature to assess stability.

Particle size of emulsions was measured using a Zetasizer Nano ZS(Malvern Instruments,) and a LA-930 Particle Distribution Analyzer(Horiba), according to the manufacturers' guidelines. Particles werediluted in deionised water. The Horiba instrument provides dl 0, d50 andd90 values i.e. the diameters which divide the droplets in the sampleinto 10%, 50% and 90% (respectively) by mass. Results were as follows:

ZETA SIZER HORRIBA Particle size Poly- d10 d50 d90 Median Mean (nm)dispersity (μm) (μm) (μm) (μm) (μm) ANE05 123.4 0.1 0.105 0.14 0.21 0.140.21 ANE06 123.5 0.1 0.109 0.16 0.31 0.16 0.59 ANE07 111.3 0.1 0.1020.14 0.21 0.14 0.47 ANE08 109.1 0.1 0.104 0.14 0.20 0.14 0.15 ANE09125.0 0.2 0.198 0.37 33.42 0.37 8.39 ANE10 117.4 0.1 0.106 0.15 0.200.15 0.15 ANE12 118.9 0.084 0.23 0.43 2.46 0.43 0.97 ANE13 — — 0.24 0.475.99 0.47 2.02 ANE14 — — 0.21 0.39 41.50 0.39 8.23 ANE15 — — 0.22 8.1533.81 8.15 12.72 ANE16 121 0.139 0.12 0.17 0.29 0.17 0.23 ANE17 127.10.169 0.23 6.48 16.18 6.49 7.08 ANE18 115.8 0.131 0.20 0.32 3.55 0.321.01 ANE19 107.9 0.081 0.10 0.13 0.18 0.13 0.14 ANE20 109.3 0.115 0.110.14 0.20 0.14 0.15 ANE21 102.5 0.07 0.12 0.19 48.09 0.19 18.26 ANE22103.8 0.091 0.10 0.13 0.20 0.13 1.24 ANE23 106 0.121 — — — — — ANE24 1050.07 — — — — — ANE31 106 0.10 0.12 0.17 0.24 0.17 0.18 ANE32 123 0.300.12 0.16 0.23 0.16 0.17 ANE33 116 0.12 0.28 1.65 2.98 1.65 1.63 ANE3497 0.06 0.10 0.14 0.21 0.14 0.67 ANE35 115 0.05 0.08 0.11 0.16 0.11 0.12

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

TABLE 1 useful phospholipids DDPC1,2-Didecanoyl-sn-Glycero-3-phosphatidylcholine DEPA-NA1,2-Dierucoyl-sn-Glycero-3-Phosphate(Sodium Salt) DEPC1,2-Erucoyl-sn-Glycero-3-phosphatidylcholine DEPE1,2-Dierucoyl-sn-Glycero-3-phosphatidylethanolamine DEPG-NA1,2-Dierucoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DLOPC1,2-Linoleoyl-sn-Glycero-3-phosphatidylcholine DLPA-NA1,2-Dilauroyl-sn-Glycero-3-Phosphate(Sodium Salt) DLPC1,2-Dilauroyl-sn-Glycero-3-phosphatidylcholine DLPE1,2-Dilauroyl-sn-Glycero-3-phosphatidylethanolamine DLPG-NA1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) (SodiumSalt) DLPG-NH4 1,2-Dilauroyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol .. .) DLPS-NA 1,2-Dilauroyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)DMPA-NA 1,2-Diimyristoyl-sn-Glycero-3-Phosphate(Sodium Salt) DMPC1,2-Dimyristoyl-sn-Glycero-3-phosphatidylcholine DMPE1,2-Dimyristoyl-sn-Glycero-3-phosphatidylethanolamine DMPG-NA1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DMPG-NH41,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .)DMPG-NH4/NA 1,2-Myristoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . ..) DMPS-NA 1,2-Dimynstoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)DOPA-NA 1,2-Dioleoyl-sn-Glycero-3-Phosphate(Sodium Salt) DOPC1,2-Dioleoyl-sn-Glycero-3-phosphatidylcholine DOPE1,2-Dioleoyl-sn-Glycero-3-phosphatidylethanolamine DOPG-NA1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DOPS-NA1,2-Dioleoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt) DPPA-NA1,2-Dipalmitoyl-sn-Glycero-3-Phosphate(Sodium Salt) DPPC1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylcholine DPPE1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylethanolamine DPPG-NA1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .)DPPG-NH4 1,2-Dipalmitoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . ..) DPPS-NA 1,2-Dipalmitoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt)DPyPE 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine DSPA-NA1,2-Distearoyl-sn-Glycero-3-Phosphate(Sodium Salt) DSPC1,2-Distearoyl-sn-Glycero-3-phosphatidylcholine DSPE1,2-Diostearpyl-sn-Glycero-3-phosphatidylethanolamine DSPG-NA1,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DSPG-NH41,2-Distearoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol . . .) DSPS-NA1,2-Distearoyl-sn-Glycero-3-phosphatidylserine(Sodium Salt) EPC Egg-PCHEPC Hydrogenated Egg PC HSPC High purity Hydrogenated Soy PC HSPCHydrogenated Soy PC LYSOPC MYRISTIC1-Myristoyl-sn-Glycero-3-phosphatidylcholine LYSOPC PALMITIC1-Palmitoyl-sn-Glycero-3-phosphatidylcholine LYSOPC STEARIC1-Stearoyl-sn-Glycero-3-phosphatidylcholine Milk Sphingomyelin MPPC1-Myristoyl,2-palmitoyl-sn-Glycero 3-phosphatidylcholine MSPC1-Myristoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine PMPC1-Palmitoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine POPC1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine POPE1-Palmitoyl-2-oleoyl-sn-Glycero-3-phosphatidylethanolamine POPG-NA1,2-Dioleoyl-sn-Glycero-3[Phosphatidyl-rac-(1-glycerol) . . .](SodiumSalt) PSPC 1-Palmitoyl,2-stearoyl-sn-Glycero-3-phosphatidylcholine SMPC1-Stearoyl,2-myristoyl-sn-Glycero-3-phosphatidylcholine SOPC1-Stearoyl,2-oleoyl-sn-Glycero-3-phosphatidylcholine SPPC1-Stearoyl,2-palmitoyl-sn-Glycero-3-phosphatidylcholine

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The invention claimed is:
 1. An oil-in-water emulsion comprising anaqueous phase, an oil phase, a surfactant, a crystallisation inhibitor,a phospholipid and a naphthyridine TLR agonist, wherein the averagedroplet size in the emulsion is less than 250 nm, the naphthyridine TLRagonist has a molecular weight of less than 5000 Da and is present at aconcentration sufficient to crystallize absent the crystallizationinhibitor, the crystallization inhibitor is a polyvinylpyrrolidone at aconcentration sufficient to inhibit crystallization of the naphthyridineTLR agonist, the phospholipid is DSPC, and >80% of the naphthyridine TLRagonist is present in the emulsion's oil droplets.
 2. The emulsion ofclaim 1, wherein the oil phase comprises squalene.
 3. The emulsion ofclaim 1, wherein the aqueous phase comprises a buffer.
 4. The emulsionof claim 1, wherein the surfactant comprises polysorbate
 80. 5. Theemulsion of claim 1, comprising 2-20% (by volume) oil and 0.001%-8% (byweight) surfactant.
 6. The emulsion of claim 1, wherein the weight ratioof oil to surfactant is between 4:1 and 5:1.
 7. The emulsion of claim 1,comprising squalene and polysorbate 80, wherein the average diameter ofthe emulsion's droplets is between 80 nm and 180 nm.
 8. An immunogeniccomposition comprising (a) the emulsion of claim 1 and (b) an immunogen.9. The composition of claim 8, wherein the immunogen elicits an immuneresponse in vivo against a bacterium or a virus.
 10. The composition ofclaim 9, wherein the virus is an influenza virus.
 11. The emulsion ofclaim 1, wherein the naphthyridine TLR agonist is a TLR7 agonist. 12.The emulsion of claim 1, wherein >90% of the naphthyridine TLR agonistis present in the emulsion's oil droplets.